The present application is the national stage under 35 U.S.C. 371 of PCT/IT97/00283, filed Nov. 18, 1997.
The subject of the present invention is the use of molecules that activate the CNTF (ciliary neurotrophic factor) receptorxe2x80x94such as hCNTF (human CNTF) or mutants of hCNTFxe2x80x94as active principles in the formulation of pharmaceutical compositions suitable for the treatment of obesity and of related diseases. The term hCNTF mutant is intended to mean an amino acid sequence that can in theory be derived from hCNTF by substitution of one or more amino acids.
Obesity, which affects  greater than 30% of the adult population in the industrial world, is a major public health problem, since it is associated with type II diabetes, hypertension, hyperlipidemia and increased mortality rate. Obesity is the result of a positive energy balance, as a consequence of an increased ratio of caloric intake to energy expenditure. Treatment is generally unsuccessful due to the operation of mechanisms that restore adipose mass after both intentional or unintentional changes (1). The lipostasis theory postulates that the size of the body fat depot is regulated by a feedback loop, constituted by adipocyte-derived circulating molecules that act on the hypothalamus to decrease appetite and increase energy expenditure (2).
The recently identified 16-kilodalton plasma protein leptin (3) fulfills many of the criteria expected from such a lipostatic hormone. It is expressed in adipose tissue, and its plasma levels are highly correlated with body mass index in rodents and humans (4). The absence of leptin in obese (ob/ob) mutant mice leads to a massive increase in body fat, which can be reversed by systemic administration of the recombinant protein (5, 6, 7). However, human obesity does not appear to be due to deficient expression of leptin, since leptin mRNA and plasma protein levels were shown to be increased in obese versus lean subjects (4). Thus, obese humans may be insensitive to the lipostatic effect of leptin, possibly due to a defect at the level of leptin transport, leptin receptor activity, or post-receptorial signalling mechanisms (8).
There is thus a need in this specific field for new pharmacological agents capable of correcting obesity in people who are resistant to leptin.
Leptin resistance is a characteristic feature of the diabetic (db/db) mouse mutant, which expresses a truncated form of the leptin receptor lacking most of the intracytoplasmic domain (9). An animal model that more closely resembles human obesity is that of mice rendered obese by feeding a high-fat diet (DIO mice). Similar to human obese subjects, DIO mice have elevated plasma levels of leptin (4), suggesting that they are relatively insensitive to the weight-reducing effects of the hormone.
The present invention provides biologically active anti-obesity agents that can reverse obesity, as well as hyperglycemia and hyperinsulinemia associated therewith.
The subject of the present invention is therefore the use of substances that activate the CNTF receptor for the preparation of drugs for treatment of obesity and related diseases. These substances can be hCNTF (human ciliary neurotrophic factor; SEQ ID NO: 1) itself or mutants thereof (see for instance SEQ ID NOS:2 to 28). Good results have been obtained using the hCNTF mutant (Ser166Asp/Gln167His) hCNTF (10), which, from position 159 to position 178, has the following amino acid sequence (shown as SEQ ID NO: 5 in the annexed sequence listing):
Leu Lys Val Leu Gln Glu Leu Asp His Trp Thr Val Arg Ser Ile His Asp Leu Arg Phe [for sake of simplicity, this hCNTF mutant will be referred to hereinafter also as DH-CNTF]. For sake of simplicity, in the annexed sequence listing, it has been indicated only the portion from position 159 to position 178 of the mutants SEQ ID NOS: 2 to 22.
A further subject of the invention is the use of DNA coding for hCNTF or mutants thereof for the preparation of compositions for the treatment of obesity and diseases related thereto.
The present invention also has as its subject a drug for the treatment of obesity and the reduction of body weight, containing, as at least one of its active principles, hCNTF or a mutant thereof, and comprising a pharmaceutically acceptable vehicle. A pharmaceutically acceptable vehicle is intended to be a vehicle that is not dangerous for the patient, that does not degrade or deactivate the active principles or that does not interfere with the effects thereof. The preferred vehicle is a physiological saline solution, but other pharmaceutically acceptable vehicles can be used, and will easily be identified by those skilled in the art. In an embodiment that has shown good results hCNTF or mutants thereof can be used in combination with leptin: in this case the ratio wild type or mutant CNTF/leptin can be selected in the range 1:500 to 1:5, preferably 1:100 to 1:25.
hCNTF or hCNTF variants can be administered to patients in need of treatment in doses ranging from about 1 to 10,000 xcexcg/kg body weight. A preferred dose is between 10 and 1000 xcexc/kg body weight. A typical daily dose for an adult is between 1 and 100 mg. The necessary amount of active principle according to the invention can be administered in a single daily dose or in multiple doses throughout the day. The treatment regime can require administration for prolonged periods. The size of the dose administered must be determined by a physician and will depend on a number of factors, such as the nature and gravity of the disease, the age and state of health of the patient and the patient""s tolerance to the drug itself.
In a specific embodiment, hCNTF or a mutant thereof can be used for treatment of obese patients by means of a short-term (1-2 weeks) daily administration, in order to obtain a rapid, significant decrease in body weight (5-10%), which can be maintained subsequently using an appropriate diet and/or physical exercise.
The active protein molecules can be formulated for parenteral, nasal, bronchial or transdermal administration. The pharmaceutical composition according to the present invention is preferably administered parenterally by means of an injection. In the preferred embodiment, parenteral administration is subcutaneous or intramuscular. Other effective methods of administration are intravenous injections, slow-release parenteral formulations, inhalant mists, or suppositories. In the slow-release formulation the primary solvent can be either of an aqueous or of a non-aqueous type. Furthermore, the vehicle can contain other pharmacologically acceptable excipients to maintain or modify the pH, viscosity, clarity, colour, sterility, stability, speed of dissolution or odor of the formulation. Similarly, the vehicle can also contain other pharmacologically acceptable excipients to modify or maintain the stability, speed of dissolution, release, or absorption of the active principle. These excipients are substances that are normally used to formulate doses for parenteral administration, both in the form of single doses and in the form of multiple doses.
As mentioned above, the preferred parenteral form of administration of the formulation according to the invention is subcutaneous or intramuscular. The most preferred form of parenteral administration is subcutaneous. To obtain the required daily dose of active principle, it is possible to resort to single or repeated subcutaneous or intramuscular injections. In a preferred embodiment of the invention, the dose of active principle is between 10 and 1000 xcexcg/kg/day. For the treatment of obesity, it may be desirable to administer the active principle periodically. Periodic administration may take the form of monthly, bi-weekly, weekly, daily or hourly administration. The required frequency of administration will be apparent to those treating the patient on the basis of standard observational techniques.
It is also possible to consider oral administration of the pharmaceutical formulations according to the invention. In this case, the active principle administered is preferably encapsulated. The encapsulated active principle can be formulated with or without the vehicles usually employed in the preparation of solid doses. Preferably, the capsule is made in such a way that the active portion of the formulation is released in the gastro-intestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. The formulation can also include further excipients with the aim of facilitating absorption of the active principle. It is also possible to use diluting agents, flavouring, low melting-point waxes, vegetable oils, lubricants, suspending agents, capsule disintegration agents and binding agents.
Independently of the method of administration, the specific dose is calculated according to the approximate body weight of the patient. Further refinement of the calculations necessary to determine the appropriate dose for treatment is routinely made by those of ordinary skill in the art, who are capable of reaching these results without the need for undue experimentation, especially in the light of the tests and dosing information provided herein.
According to the present invention, an obese patient is administered a therapeutically effective amount of active principle. As mentioned above, the dose required can be determined by those skilled in the art without the need for undue experimentation. A xe2x80x9ctherapeutically effective amountxe2x80x9d can be defined as the amount of active principle that is sufficient to cause an adequate loss of weight and to result in the consequent normalisation of metabolic parameters, such as the blood glucose level of the obese patient.
Up to this point a general description has been given of the present invention. With the aid of the following examples, a more detailed description will now be provided, with reference to specific embodiments, aimed at giving a better understanding of the aims, characteristics, advantages and operating methods of the invention. However, the scope of the present invention is not intended to be limited thereby.